It is known to induce a chemiluminescent reaction between a signal reagent and a liquid analyte such as TSH, by conducting the following steps in a container with walls bearing a coating of avidin:
a) positioning the metering tip a predetermined distance above the upper level of the liquid analyte prior to dispensing a labeled liquid reagent specific to the TSH, that is, an antibody to the TSH labeled with horse-radish peroxidase (hereinafter, HRP); PA1 b) while maintaining the distance throughout the dispensing of the liquid reagent, dispensing that reagent; PA1 c) repeating steps a) and b) for a second liquid reagent, for example one capable of reacting with both the TSH and the container wall, e.g., an antibody to TSH that is bound to biotin; PA1 d) incubating the liquids present in the container for a time sufficient to bind the labeled reagent to any of the TSH present in the sample, and the TSH to the container; PA1 e) washing out unbound amounts of the labeled reagent from the container by i) removing the liquids therefrom, ii) adding wash fluid back to the container, and iii) repeating steps i) and ii) at least once; and PA1 f) adding the signal reagent to induce chemiluminescence. The known process uses dispensing heights (the predetermined distances) that range from 8.34 mm at the maximum to 5.58 mm at the minimum. PA1 a) positioning the metering tip a predetermined distance above the upper level of the second liquid prior to dispensing the one liquid; and PA1 b) while maintaining the distance throughout the dispensing of the one liquid, dispensing said one liquid; PA1 wherein the predetermined distance is between about 1.0 mm and about 2.0 mm so that splashing during dispensing is reduced. PA1 a) dispensing from a tip an amount of the labeled analyte-specific reagent for delivery into the sample liquid in the container, while maintaining throughout the dispensing, the height of the tip from the sample liquid a first predetermined distance; PA1 b) optionally adding on additional reagent to the sample liquid intermixed in the container with the liquid reagent added in step b), by dispensing from a tip the optional additional reagent all while maintaining the height of the tip from the liquid in the container at a second predetermined distance, at least one of the reagents being capable of binding to the container; PA1 c) incubating the liquids present in the container for a time sufficient to bind the labeled reagent to any of the TSH present in the sample, and the TSH to the container; PA1 d) washing out unbound amounts of the label reagent from the container by i) removing the liquids therefrom, ii) adding wash fluid back to the container, and iii) repeating steps i) and ii) at least once; PA1 e) adding a signal reagent to the container effective to react with the bound labeled reagent to produce a detectable result; and PA1 f) detecting the result; PA1 wherein the predetermined maintained distances are reduced to a value of from between about 1.0 mm to about 2.0 mm, so that splashing of the labeled reagent out of the liquid onto the container walls where it becomes irretrievably bound during step c), is reduced.
This process works generally quite well with most analytes. However, with TSH, it has been discovered that outliers can result that produce a positive bias. Because the containers are thoroughly washed to remove free labeled reagent, that is, reagent unbound through the analyte to the container, it was not at all clear what the cause was for this problem. Extensive modifications were attempted without success.
As used herein, "unbound" reagent refers to reagent that is not immobilized to the container through a conventional attachment process, such as a sandwich assay involving the analyte and an immobilized analyte-specific reagent. Such unbound reagent can be complexed with analyte not complexed with the immobilizing analyte-specific reagent. In this latter case, the complex of label reagent and analyte is conventionally removed by the wash process--hence it is considered "unbound".
Thus, there has been a need for a solution to the positive bias occasionally produced in TSH analysis.